Stressed out about RNA Granules; Roy Parker

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Live blogger: Whit Froehlich. Editor: Sarah Kearns.

Roy Parker, Ph.D., is a Professor at the University of Colorado and Investigator at the Howard Hughes Medical Institute who studies the regulation of translation and degradation of messenger RNA (mRNA) using yeast as a model organism. Degradation of mRNA is accomplished in coordination with the removal of the “poly-A tail” at its end, which precedes degradation from either end of the molecule. His other work includes investigation of the processes around mRNA decapping and storage in P-bodies. He is speaking today about RNA granules, and generally about aggregated RNA structures, as well as some of their roles in disease.

Firstly, RNA granules, aggregations of protein and RNA, come in a number of different forms including Cajal bodies in the nucleus, and P-bodies and stress granules in the cytoplasm. Stress granules are assemblies of non-translating mRNAs with a number of the enzymes normally involved in translation and processing of proteins. These granules have a role in transitions between different states of mRNAs, assembly of organelles, and disease processes.

One example is dendrites of neurons, where mRNA is first stored in granules and then later translated in certain circumstances. This process is important for synaptic plasticity and habit formation. An experiment around this function involved observing first that flies become habituated to an unpleasant scent, and then removing the function of Atx2, an important regulator protein. This removed their ability to habituate, and so they no longer would eventually ignore the unpleasant odor. Furthermore, they no longer accumulated the RNA granules seen in the normal flies suggesting the importance of the granules in habit formation. The genetic domains were identified which coded for the granule proteins, where the deletion of just these domains had the same effect on fly habituation, by inhibiting it.

Granules also play a role in development, regeneration, stress responses, and neurodegenerative disease. It has been observed that mutations causing neurodegenerative disease often alter the presence of RNA stress granules. It turns out that they code for RNA binding proteins. Likewise, genes coding for complexes that disassemble stress granules, when mutated, can correlate with the development of neurodegenerative disease.

Stress granules have been purified, and the proteins in them identified. Less is known about their RNA content, however. In analyzing the RNAs that appear in stress granules, there is a continuum in the range of genes that are expressed at higher, lower, or similar levels to elsewhere in the cell. This invited the suggestion that perhaps the sequencing was inaccurate; but single molecule validation performed on stress granules confirmed correct localization. So, back to the stress granules: it turns out that only about 10-15% of mRNAs appear in stress granules, but none particularly predominate, although there are a few genes for which most of their transcribed mRNA ends up in stress granules.

Looking at the different types of RNA, longer mRNA sequences only 10% of mRNA ends up in stress granules with longer, poorly translated sequences forming the bulk of the granule. Image Source.

The genes that are related to the mRNA within granules have two identified properties: poor translation and length (~7.5kb). As to the latter, it was considered that perhaps length itself, and the correspondingly large amount of protein-binding site, was the predisposing factor. However, enrichment with binding protein found that this was not actually important. On the other hand, a longer mRNA also has a greater opportunity to bind with itself. An experiment performed with roughly cell-like quantities of RNA in the absence of granule proteins found that they still become entangled nonetheless. Analysis of the RNAs that tended to self-bind discovered that these were the same RNAs that assemble in stress granules. The suggestion from these various findings is that protein-protein interactions and RNA-RNA interactions, rather than protein-RNA interactions, drive stress granule formation.

Going back to proteins, it is found that while stress granules can form with either mostly RNA or mostly protein, the formation is still limited by inhibition of either. Notably, RNA tend to be more or less likely to self-aggregate depending on what type of RNA they are: tRNAs are not observed to clump together, while longer strands encoding genetic sequences with many repeats will. In general, the more structured an RNA molecule is in its functional state, the less likely it is to self-aggregate.

Other aspects of the environment affect the tendency of RNA to form granules: crowding, length, and the absence of RNA binding proteins all make granule formation more likely. Dr. Parker even suggests that, without protein complexes, the lowest-energy state of RNA might still be in forms that promote aggregation. Based off of that, another factor that affects granule formation is the presence of polyamines (amino acid clusters of arginine and glycine or proline), which drive RNA self-assembly. These polyamines, because of their positive charge, interact with the negative charges on RNA and prevent RNA granule formation. The effect of polyamines is paralleled by the deletion of the domains found to encode granule proteins playing a role in habituation; deletion of the same domains also inhibited necrosis of fly eyes caused by a similar process.